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Title: Permeation and metabolism of intermediates of the mandelate pathway in bacterium NCIB 8250
Author: Cook, Alasdair Macleod
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 1972
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Abstract:
Many bacteria have a very wide metabolic versatility, generally in the form of convergent, inducible, catabolic pathways for the enzymic degradation of organic compounds. The regulation of these pathways is poorly understood. Theories on enzyme regulation are built on the operon model for the control of gene expression and the concept of allosteric control, of enzyme activity, while the permeability of the cell membrane has also been considered as a possible locus to mediate enzyme regulation. The mandelate pathway, of an Acinetobacter species, a soil organism known as bacterium NCIB 8250, is one defined unit in a convergent, inducible, catabolic sequence. The experimental work described in this thesis is concerned with the control of the enzymes converting L-mandelate and benzyl alcohol to catechol in bacterium NCIB 8250 and specifically with the role of permeation in the control mechanisms. A sensitive radiochemical assay was developed to measure the utilisation of mandelate, benzyl alcohol or benzoato during growth of bacterium NCIB 8250 in batch culture. An exponential growth curve was observed when either benzoate or benzyl alcohol was the sole source of carbon and energy. Growth in mandelate-salts medium was complex and involved the exhaustion of mandelate before the end of growth a transient accumulation of benzaldehyde and sub-sequent growth on excreted material. Growth in dual substrate' media was examined and certain cases of the preferential use of one substrate were observed. The utilisation of mandelate, use of one substrate were observed. The utilization of mandelate was supressed when benzoate, catechol or succinate was present, but not when benzyl alcohol was present. Suppression of the utilisation of benzyl alcohol in the presence of mandelate occurred after a delay, while benzoate suppressed benzyl alcohol utilization in cells preinduced to growth on benzoate but not in cells preinduced to growth on benzyl alcohol. No suppression of the utilization of benzoate was observed. The use of cell-free enzyme assays in the other intermediates showed that enzyme inhibitions were not the cause of the suppressions of substrate utilization. In whole cells, the utilization of mandelate, benzyl alcohol or benzoate was generally unaffected by other intermediates though catechol and succinate did have inhibitory effects (about 30%) of unknown origin. The utilization of mandelate in cells possessing the mandelate enzymes was not inhibited by benzoate, so suppression of mandelate utilization in the presence of benzoate was ascribed to repression of protein synthesis. At least some part of the effects of succinate and catechol in suppressing the utilization of mandelate was also ascribed to repression of protein synthesis. The enzymes for the utilization of benzyl alcohol were represented by L-mandelate, but no repression by benzoate catechol or succinate was detected. No repression of benzoate oxidase was detected. A radiochemical assay to measure very low quantities of intracellular aromatic intermediate was developed. A general permeability barrier to carbohydrate was exploited to estimate the intracellular water space and intracellular material was expressed as a concentration in that same water space. Varying degrees of permeability barriers to mandelate were displayed by cells without L-mandelate dehydrogenase. Rapid decarboxylation of mandelate was observed in induced wild type cells. An inducible mandelate transport system was therefore proposed. A high radiochemical reagent blank for benzyl alcohol prevented use of the radiochemical assay to study the entry of benzyl alcohol into cells in suspension. However, an inducible benzyl alcohol transport system was proposed on indirect evidence from growth experiments: benzoate suppressed the utilization of benzyl alcohol in cells preinduced to growth on benzoate without inhibition or repression in cells preinduced to growth on benzyl alcohol. No permeability barrier to benzoate itself was observed, and no substantial inhibition of the decarboxylation of benzoate by whole cells was detected in the presence of a wide range of analogues of benzoate. One of these compouds, p-fluoro-benzoato was decarbozylated by suspensions of cells induced to growth on benzoate. This decarboxylation was totally prevented by the presence of benzoate. Accordingly, a constitutive benzoate transport system was postulated. It thus appears that although a permeability barrier can prevent enzyme induction by preventing entry of inducer into the cell, repression of enzyme synthesis in the mandelate pathway of bacterium NCIB 8250 is a control at the level of the protein synthesis that is not mediated by an inhibition of transport. The possible relevance of the transport systems which have been postulated in this organism and the controls, in terms of multisensitive, end-product repression, are discussed.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.776365  DOI: Not available
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